The present invention is directed to nucleic acid molecules encoding Polistinae venom allergens, in particular enzymes such as phospholipase and hyaluronidase, or fragments thereof, recombinant vectors comprising such nucleic acid molecules, and host cells containing the recombinant vectors. The invention is further directed to expression of such nucleic acid molecules to produce a recombinant Polistinae venom enzyme, such as phospholipase or hyaluronidase, or recombinant fragments thereof. Such an allergen and fragments thereof are useful for diagnosis of allergy, for therapeutic treatment of allergy, for the treatment of immune system related diseases or disorders, or symptoms related thereto, and for the modulation of immune response towards an immunogen.
Insect sting allergy to bees and vespids is of common occurrence. The vespids include hornets, yellow jackets and wasps (Golden, et al., 1989, Am. Med. Assoc. 262:240). Susceptible people can be sensitized on exposure to minute amounts of venom proteins; as little as 2-10 xcexcg of protein is injected into the skin on a single sting by a vespid (Hoffman and Jacobson, 1984, Ann. Allergy. 52:276).
There are many species of hornets (genus Dolichovespula), yellow jackets (genus Vespula) and wasp (genus Polistes) in North America (Akre, et al., 1980, xe2x80x9cYellowjackets of America North of Mexico,xe2x80x9d Agriculture Handbook No. 552, US Department of Agriculture). The vespids have similar venom compositions (King, et al., 1978, Biochemistry 17:5165; King, et al., 1983, Mol. Immunol. 20:297; King, et al., 1984, Arch. Biochem. Biophys. 230:1; King, et al., 1985, J. Allergy and Clin. Immunol. 75:621; King, 1987, J. Allergy Clin. Immunol. 79:113; Hoffman, 1985, J. Allergy and Clin. Immunol. 75:611). Their venom each contains three major venom allergens, phospholipase (37 kD), hyaluronidase (43 kD) and antigen 5 (23 kD) of as yet unknown biologic function. U.S. Pat. No. 5,593,877 describes cloning and expression of the vespid venom allergens phospholipase and hyaluronidase. As described in this patent, the recombinant allergens permit expression of a protein or fragments thereof for use in immunotherapy, dignostics, and to investigate T and B cell allergens, it sets forth in greater detail the rationale for cloning vespid venom enzymes. However, unique vespid venom cDNAs were not described.
In addition to the insect venom allergens described above, the complete amino acid sequence of several major allergens from different grass (Perez, et al., 1990, J. Biol. Chem. 265:16210; Ansari, et al., 1989, Biochemistry 26:8665; Silvanovich, et al., 1991, J. Biol. Chem. 266:1204), tree pollen (Breiteneder, 1989, EMBO J. 8:1935; Valenta, et al., 1991, Science, 253:557), weed pollen (Rafnar, et al., 1991, J. Biol. Chem. 266:1229; Griffith, et al., 1991, Int. Arch. Allergy Appl. Immunol. 96:296), mites (Chua, et al., 1988, J. Exp. Med. 167:175), cat dander (Griffith, et al., 1992, Gene. 113:263), and mold (Aruda, et al., 1990, J. Exp. Med. 172:1529; Han, et al., 1991, J. Allergy Clin. Immunol. 87:327) have been reported in the past few years. These major allergens are proteins of 10-40 kD and they have widely different biological functions. Nearly all allergens of known sequences have a varying extent of sequence similarity with other proteins in our environment.
Although U.S. Pat. No. 5,593,877 provides for cloning and expression of vespid venom enzymes, particularly hyaluronidase and phospholipase, there remains a need to identify unusual and unexpected sequences for such enzymes, and to design effective expression systems for them. There is a particular need to delineate the B and helper T cell epitopes of the paper wasp (e.g., Polistes annularis). In particular, the major Polistinae venom allergens phospholipase and hyaluronidase are appropriate targets for determining the important B and T cell epitopes. In order to fully address the basis for allergic response to vespid allergens, and to develop allergen-based immunotherapies, the cDNA and protein sequences of several homologous allergens need to be investigated. Moreover, vectors suitable for high level expression in bacteria and eukaryotic cells of vespid allergens or their fragments should be developed. Recombinant vespid allergens and their fragments may then be used to map their B and T cell epitopes in the murine and, more importantly, human systems by antibody binding and T cell proliferation tests, respectively.
There is also a need in the art to use peptides having T or B cell epitopes of vespid venom allergens to study induction of tolerance in mice and induction of tolerance in humans.
There is a further need to test whether a modified peptide inhibits allergen T cell epitope binding to MHC class II molecule, or induces T cell anergy, or both.
Thus, there is a need in the art for unique sequence information about vespid venom allergens, and a plentiful source of such allergens for immunological investigations and for immunological therapy of the allergy.
Furthermore, due to the overuse of antibiotics throughout the world, and to the spread of numerous viruses, such as HIV, Ebolla, etc., efforts have been made to produce new xe2x80x9csuperxe2x80x9d antibiotic medication, and compounds which have activity against viruses. For example, AZT has been developed, along with protease inhibitors to treat subjects suffering from HIV. However, the costs of developing new xe2x80x9csuperxe2x80x9d antibiotics and anti-viral medications are enormous.
Hence, what is needed are agents and pharmaceutical compositions for treating immune system related diseases or disorders whose activity is not dependent necessarily on combating the particular virus or pathogen, but rather modulate or potentiate the immune system ability to combat the disease or disorder, thereby ameliorating the disease or disorder, or a symptom related thereto. Hymenoptera venoms, particularly vespid venoms, provide one possible source for such agents and pharmaceutical compositions, as described in U.S. Pat. Nos. 4,822,608 and 5,827,829.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.
The present invention provides a nucleic acid molecule encoding Polistinae venom enzymes, immunomodulatory fragments thereof, or derivatives or analogs thereof. In particular, the invention is directed to such nucleic acid molecules encoding a Polistinae venom phospholipase, and a Polistinae venom hyaluronidase. In specific embodiments, a nucleic acid molecule of the invention encodes an immunomodulatory portion of a T cell epitope of a Polistinae venom enzyme. In another embodiment, a nucleic acid molecule of the invention encodes an antigenic portion of a B cell epitope of a Polistinae venom enzyme.
The nucleic acids of the invention, which are not genomic, surprisingly are found, in one embodiment, to contain a non-coding, e.g., intronic sequences. In a specific embodiment, cDNA molecules for Polistinae venom enzyme contain what appears to be an intron. Thus, it has unexpectedly proved necessary to delete these xe2x80x9cintronicxe2x80x9d sequences in order to obtain a nucleic acid coding for a mature Polistinae venom enzyme, e.g., phosholipase or hyaluronidase.
Hence broadly, the present invention extends to an isolated nucleic acid molecule encoding a venom enzyme, conserved variant thereof, immunomodulatory fragment thereof, or derivative, or analog thereof. As noted above, the nucleic acid molecule contains internal non-coding sequences, i.e., in addition to 5xe2x88x92 and 3xe2x88x92 untranslated (UTR) sequences., but is not a genomic sequence. Examples of Polistinae venom enzymes which can be encoded by an isolated nucleic acid molecule of the invention include, but are not limited to phospholipase and hyaluronidase. Moreover, enzymes, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof, from the venom of numerous Polistinae venoms can be encoded by an isolated nucleic acid molecule of the invention. A particular example comprises Polistinae of the genus Polistes, and particularly the species annularis. 
In a particular embodiment, the present invention extends to an isolated nucleic acid molecule encoding a phospholipase A1, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof, from the genus Polistes and the species annularis, wherein the P. annularis has an amino acid sequence as depicted in SEQ ID NO:2, and more specifically, wherein the isolated nucleic acid molecule has a nucleotide sequence of SEQ ID NO: 1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
In another particular embodiment, the present invention extends to an isolated nucleic acid molecule, that encodes hyaluronidase from Polistes annularis comprising an amino acid sequence of SEQ ID NO: 4, more particularly wherein the isolated nucleic acid has a nucleotide sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof.
Moreover, the present invention extends to an isolated nucleic acid molecule hybridizable to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1 or 3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Moreover, the present invention further extends to an isolated nucleic acid molecule encoding a Polistinae venom enzyme, or an immunomodulatory fragment, derivative or analog thereof, wherein the isolated nucleic acid molecule encodes an immunomodulatory portion of a T cell epitope or an antigenic portion of a B cell epitope of the Polistinae venom enzyme. Likewise, the present invention extends to an isolated polypeptide comprising an immunomodulatory portion of a T cell epitope of a Polistinae venom enzyme, wherein the polypeptide is encoded by an isolated nucleic acid molecule of the invention. Examples of waso venom enzymes for which isolated nucleic acid molecules of the present invention encode an immunomodulatory portion of a T cell epitope include, but certainly are not limited to, phospholipase and hyaluronidase. In a specific embodiment, the phospholipase A1 and hyaluronidase originate from a genus Polistes, and particularly from the species annularis. 
The invention further provides cloning vectors and expression vectors, which permit expression of the nucleic acids. Such vectors contain nucleic acids of the invention as set forth above. In the case of expression vectors, such nucleic acids are operatively associated with an expression control sequence.
The invention advantageously provides a method of producing a Polistinae venom phospholipase, conserved variant thereof, immunomodulatory fragment thereof, or analog or derivative thereof, which is encompassed by the present invention, comprises:
(a) culturing a host cell transformed with an expression vector comprising an isolated nucleic acid molecule hybridizable to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO: 1, preferably having a sequence of SEQ ID NO: 1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, wherein the isolated nucleic acid molecule is operationally associated with a promoter, so that the Polistinae venom phospholipase, conserved variant thereof, immunomodulatory fragment thereof, or analog or derivative thereof, is produced by the host cell; and
(b) recovering the Polistinae venom phospholipase, conserved variant thereof, immunomodulatory fragment thereof, or analog or derivative thereof so produced from the culture, the host cell, or both.
Another method is provided for producing a Polistinae venom hyaluronidase, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof, comprises the steps of:
(a) culturing a host cell transformed with an expression vector comprising an isolated nucleic acid molecule hybridizable to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3, or preferably having a sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, wherein the isolated nucleic acid molecule is operationally associated with a promoter, so that the Polistinae venom hyaluronidase, conserved variant thereof, immunomodulatory fragment thereof, or analog or derivative thereof is produced by the host cell, and
(b) recovering the Polistinae venom hyaluronidase, conserved variant thereof, immunomodulatory fragment thereof, or analog or derivative thereof so produced, from the culture, the host cell, or both.
In a particular example, the methods set forth above yield phospholipase A1 or hyaluronidase of the genus Polistes, and particularly from the species annularis, wherein the phospholipase A1 comprises an amino acid sequence of SEQ ID NO:2, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof, and the hyaluronidase comprises an amino acid sequence of SEQ ID NO:4, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof.
The present invention further extends to pharmaceutical compositions effective for the treatment of a venom allergen-specific allergic condition. In particular, the present invention extends to a pharmaceutical composition comprising a polypeptide encoded by an isolated nucleic acid molecule which encodes an immunomodulatory portion of a T cell or an antigenic portion of a B cell epitope of a Polistinae venom enzyme, e.g., phospholipase or hyaluronidase, and a pharmaceutically acceptable carrier thereof. Consequently, in a preferred embodiment, a pharmaceutical composition of the invention comprises an immunomodulatory T cell epitope of Polistes annularis venom phospholipase A1, or hyaluronidase or an antigenic portion of a B cell epitope of Polistes annularis phospholipase A1, or hyaluronidase.
Naturally, the present invention extends to a method for treating a vespid venom allergen-specific allergic condition comprising administering a therapeutically effective amount of a pharmaceutical composition of the invention, examples of which are set forth above. Administration of a pharmaceutical composition of the invention can occur parenterally, and particularly orally, pulmonarily, nasally, topically or systemically.
Furthermore, the present invention extends to use of a recombinant Polistinae venom enzyme of the invention in the manufacture of a medicament for, and an associated method for modulating an immune response towards an immunogen, e.g., treating a vespid allergic condition or treating an immune system related disease or disorder or a symptom of the immune system related disease or disorder. The polypeptide is encoded by an isolated nucleic acid molecule which encodes a Polistinae venom enzyme, wherein the polypeptide comprises an immunomodulatory fragment of a Polistinae venom enzyme. More particularly, an agent for treating an immune system related disease or disorder, or symptom related thereto, comprises a Polistinae venom enzyme or a vector that permits expression of the Polistinae venom or enzyme in vivo.
In a specific embodiment, the polypeptide is a phospholipase encoded by an isolated nucleic acid molecule hybridizable to, or preferably, comprising a DNA sequence of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Hence, an agent for treating an immune system related disorder or disease, or a symptom thereof, comprises an isolated polypeptide encoded by an isolated nucleic acid molecule which encodes a Polistinae venom hyaluronidase, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof.
In another embodiment, the polypeptide is a hyaluronidase encoded by an isolated nucleic acid molecule hybridizable to, and preferably comprising, a DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Furthermore, the present invention extends to a pharmaceutical composition for modulating an immune response towards an immunogen, e.g., treating a vespid allergic condition or treating an immune system related disease or disorder or a symptom related thereto, wherein the pharmaceutical composition comprises a recombinant Polistinae venom enzyme and a pharmaceutically acceptable carrier thereof.
Administration of a pharmaceutical composition for treating an immune system related disease or disorder to a subject can be carried out parenterally, and particularly orally, pulmonarily, nasally, topically or systemically. Furthermore, numerous diseases or disorders related to the immune system can be treated with the present invention. Examples include, but are no limited to, a pathogenic disease or disorder such as a viral disease or disorder, e.g., HIV, Herpes Simplex virus, or papiloma virus; an autoimmune disease e.g. arthritis or Lupus; or a combination of such diseases or disorders.
It is a specific object of the invention to provide the surprising DNA sequence of isolated nucleic acid (cDNA) molecules that encode Polistes annularis hyaluronidase, conserved variants thereof, fragments thereof, or analogs or derivatives thereof.
It is still yet another object of the invention to provide amino acid sequences of Polistes annularis phospholipase A1, and hyaluronidase, along with conserved variants thereof, fragments thereof, including immunomodulatory portions of T cell epitopes and antigenic portions of B cell epitopes of Polistes annularis phospholipase A1 and hyaluronidase, either containing, or more preferably free, of xe2x80x9cintronicxe2x80x9d sequence. The deduced amino acid sequences of phospholipase A1, and hyaluronidase from Pol a allow comparison of their homology to analogous enzymes from other vespids. This information provides a basis for evaluating cross-reactivity of the allergens, which can be important for allergic reactions and for therapeutic treatments. Hence, in a specific embodiment, the present invention enables one of ordinary skill in the art to determine and evaluate the degree of similarity of phospholipase A1 and hyaluronidase of Pol a to environmental proteins and/or autologous proteins. It is believed that similarity of the vespid venom enzymes to such environmental proteins, and particularly to autologous proteins, has important implications for the allergic response.
It is yet still another object of the invention to provide expression and cloning vectors comprising an isolated nucleic acid molecule encoding Polistes annularis phospholipase A1 and hyaluronidase, including fragments comprising an immunomodulatory portion of a T cell epitope or an antigenic portion of a B cell epitope of these Polistinae venom enzymes so that the isolated nucleic acid molecules can be reproduced and expressed.
Yet another object of the invention comprises production of Polistinae venom enzymes such as phospholipase and hyaluronidase, along with conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof, using expression vectors of the invention, despite the presence of intronic sequences in cDNA clones
Yet still another object of the invention is to provide agents and pharmaceutical compositions for treating an allergen-specific allergic condition in a subject, wherein the agents and pharmaceutical composition comprise an isolated polypeptide encoded by an isolated nucleic acid molecules which encodes a Polistinae venom enzyme, such as phospholipase or hyaluronidase, particularly from Polistes annularis, wherein the polypeptide comprises an antigen portion of a B cell epitope, or an immunomodulatory portion of a T cell epitope of, a Polistinae phospholipase A1 or hyaluronidase.
Yet still another object of the invention is to provide a method for treating a vespid venom allergen-specific allergy in a subject, wherein a pharmaceutical composition for treating an allergen-specific allergic condition is administered to the subject.
Yet still another object of the invention is to provide agents and pharmaceutical compositions comprising such agents that treat an immune system related disease or disorder in mammal, such as a pathogenic disease or disorder, a viral disease or disorder, an autoimmune disease or disorder, or a combination of immune system related diseases or disorders.
Still yet another object of the invention is to provide agents and pharmaceutical composition for modulating immune response towards an immunogen in a mammal. As a result, administration of such a pharmaceutical composition modulates the immune system""s ability to recognize and attack the immunogen. In a particular embodiment, the ability of the immune system of the mammal to recognize and attack the immunogen is increased upon administration of the pharmaceutical composition relative to the ability of the subject""s immune system to recognize and attack the immunogen prior to administration of a pharmaceutical composition of the invention.